The examination of biological specimens, in which the illumination of the specimen is effected by means of a light sheet, the plane of which—the light sheet plane—intersects the optical axis of the detection—the detection direction—at an angle different from zero, has recently gained importance. The light sheet plane usually forms an angle different from zero, often—but not imperatively—a right angle, with the detection direction, which as a rule corresponds to the optical axis of the detection objective. Such examination methods are predominantly used in fluorescence microscopy and are subsumed under the term LSFM (Light Sheet Fluorescence Microscopy). An example is the method described in DE 102 57 423 A1 and WO 2004/0535558 A1, which is based thereon, and called SPIM (Selective Plane Illumination Microscopy), with which even relatively thick specimens can be spatially recorded in a relatively short time: a visually/spatially extensive representation of the specimen is possible on the basis of optical sections combined with a relative movement in a direction perpendicular to the sectional plane.
Compared with other established methods, such as confocal laser scanning microscopy or two-photon microscopy, LSFM methods have several advantages. As the detection can be effected in wide-field, larger specimen regions can be covered. Although the resolution is slightly lower than in confocal laser scanning microscopy, thicker specimens can be analysed with the LSFM technique, as the penetration depth is greater. In addition the light load on the specimen is at its lowest in this method, which reduces the risk of bleaching of a specimen as the specimen is only illuminated by a thin light sheet at an angle to the detection direction different from zero.
Instead of a purely static light sheet, a quasi-static light sheet can also be generated by rapidly scanning the specimen with a light beam. The light sheet-type illumination is formed by subjecting the light beam to a very rapid movement relative to the specimen to be observed and stringing several together sequentially over time. The integration time of the camera, onto the sensor of which the specimen is imaged, is chosen such that the scanning is completed within the integration time.
One of the main applications of light sheet microscopy is the imaging of medium-sized organisms of from several 100 μm to a few mm. As a rule these organisms are embedded in a gel, for example agarose, which is in turn located in a glass capillary tube. The glass capillary tube is introduced into a water-filled specimen chamber from above or from below and the specimen presses a piece out of the capillary. The specimen in the agarose is illuminated with a light sheet and the fluorescence is imaged onto a camera by means of a detection objective which—preferably, but not imperatively—is perpendicular to the light sheet and thus also perpendicular to the illumination objective of an optical system for generating a light sheet.
This method of light sheet microscopy, however, is subject to certain limitations. Firstly, the specimens to be examined are relatively large, they originate from developmental biology. Secondly, because of the specimen preparation and the dimensions of the specimen chamber, the light sheet is relatively thick and thus the achievable axial resolution is limited. Thirdly, the preparation of the specimens is laborious and not compatible with standard specimen preparations and standard specimen holders, such as are usual in fluorescence microscopy for individual cells.
In order to partially avoid these limitations, in recent years a novel structure has been implemented, in which the illumination objective and the detection objective are preferably perpendicular to each other and are directed onto the specimen from above at an angle of 45°. Such procedures are described, for example, in WO 2012/110488 A1 and in WO 2012/122027 A1.
As a rule coherent light of a laser is used to illuminate the specimen. In fluorescence microscopy the wavelength of the light is chosen depending on markers which are to be excited to emit fluorescence. In the simplest case, for example, a light beam with an intensity profile which corresponds to a Gaussian function can be shaped into a light sheet statically by means of cylindrical lenses, or quasi-statically by means of scanning and matched integration time of the camera. A structured illumination of the specimen, which can increase the resolution, is advantageous. Thus, for example, the coherent superimposition of Bessel beams is described in an article by V. Kettunen et al., “Propagation-invariant spot arrays”, published in Optics Letters 23(16), page 1247, 1998. The superimposition is achieved by calculating a phase element which can be introduced into the pupil with the aid of an algorithm. If the spectrum of a Bessel beam is imaged into the pupil, the phase element generates a plurality of Bessel beams which are superimposed in the specimen. The phase element is similar to a star-shaped grating with the phase values 0 and π. It is specified as a condition that the distances between the individual Bessel beams must be great as, otherwise, undesired interference effects can result.
In US 2013/0286181 A1 the interference effects between the individual Bessel beams are used in a targeted manner in order to generate an extensive and structured light sheet. Here, the Bessel beams are placed side by side so closely that the side lobes of the individual Bessel beams are destructively superimposed above and below the propagation plane, the light sheet plane. Depending on the distance of the individual Bessel beams from each other, different interference patterns result.
The generation of so-called sinc3 beams is described in WO 2014/005682 A1. An almost box-shaped light sheet with only small side lobes can thus be generated in the specimen. The sinc3 beam can be described in the frequency domain as the product of three sinc3 functions:
      f    =                  f        vr            ⁢              f        vx            ⁢              f        vy              with                              f          vr                ⁡                  (                      v            r                    )                    =              sin        ⁢                                  ⁢                  c          ⁡                      [                                                            (                                                            v                      r                                        -                                          c                      r                                                        )                                2                                            w                z                                      ]                                ,                            f          vx                ⁡                  (                      v            x                    )                    =              sin        ⁢                                  ⁢                  c          ⁡                      [                                                            (                                                            v                      x                                        -                                          c                      x                                                        )                                2                                            w                x                                      ]                                ,                  ⁢                            f          vy                ⁡                  (                      v            y                    )                    =              sin        ⁢                                  ⁢                  c          ⁡                      [                                                            (                                                            v                      y                                        -                                          c                      y                                                        )                                2                                            w                y                                      ]                                and                    v        r            ⁡              (                              v            x                    ,                      v            y                          )              =                                        v            x            2                    +                      v            y            2                              .      
The coefficients cr, cx and cy indicate the position of the sinc3 beam in the pupil plane, the coefficients wx, wy and wz indicate the width of the sinc3 beam in the respective direction.
The Fourier transform of this function f yields the complex electric field EF of the light sheet. The intensity distribution I in the focus results from I=abs(EF)2, the phase φ results at φ=arg(EF). Sinc3 beams can also be superimposed coherently, with the result that a structured, grating-type light sheet forms.
To generate the above-described beam types, for example, spatial light modulators (SLMs) can be used. For Bessel beams, for example, this is described in an article by Rohrbach et al., “A line scanned light-sheet microscope with phase shaped self-reconstructing beams”, published in Optics Express 18, page 24229 in 2010. There are two types of spatial light modulators, which differ by the liquid crystals used.
Nematic SLMs make a maximum continuously adjustable phase deviation from 0 up to 6π possible. However, these SLMs are relatively slow: as a rule, they have frame rates of about 60 Hz, at most of up to 500 Hz. In contrast the diffraction efficiency of nematic SLMs is over 90%.
On the other hand, there are ferroelectric SLMs, which can only switch back and forth between states without a phase deviation and with a phase deviation of π. For this, these SLMs are very rapidly switchable, with the result that frame rates of up to 4000 Hz can be achieved. However, the diffraction efficiency, at about 14%, is very low.
Both SLM types have in common the fact that the achievable phase deviation depends on the wavelength of the irradiated laser light. A nematic SLM should ideally be calibrated such that it has a continuous phase deviation from 0 up to an entire wavelength, thus 2π. This phase deviation can, however, only be set at a single wavelength, for which it is then designed. As soon as the SLM is irradiated with a laser of a different wavelength, the phase deviation changes and is not equal to 2π. At a shorter wavelength the phase deviation becomes larger, at a longer wavelength the phase deviation decreases.
The behaviour of a ferroelectric SLM is similar. Here too, the phase deviation of π is only achieved at the wavelength for which the pattern represented on the SLM is designed. At different wavelengths the phase deviation differs from π. This has direct effects on the generation of the above-named Bessel or sinc3 beams, thus these beams can be generated optimally only when the phase deviation continuously varies between 0 and 2π, as is necessary for Bessel and Mathieu beams, or when the phase deviation is exactly π, as is the case for coherently superimposed Bessel or sinc3 beams.
For analysis by means of fluorescence microscopy, a specimen is often prepared with different markers which can each make different regions of the specimen visible. It is therefore desirable to excite the specimen with light sheets of different wavelengths in light sheet microscopy. When a spatial light modulator is used for the beam shaping, an optimal multi-colour excitation can be implemented if the individual differently coloured light sheets are irradiated sequentially and the phase pattern is adapted to the SLM when the wavelength changes. For example, in a first pass the SLM is set to a first laser wavelength and an image stack is recorded in the z direction, the detection direction, at this wavelength. After that the SLM is set to a second wavelength and once again an image stack is recorded at this wavelength. However, this method has the disadvantage that the recording of the image stack can last a relatively long time, up to several tens of seconds. If the specimen moves or changes during this time, the differently coloured image stacks no longer match and cannot be combined into a total image.
Alternatively, each individual image of the image stack can first be recorded with the different wavelengths before the next image of an image stack is recorded. However, this has the disadvantage that with illumination times of about 10 ms the optimization of the spatial light modulator to the new laser wavelength must take place about 100 times a second. With normal nematic SLMs this cannot be achieved, with the result that ferroelectric SLMs, with their low light efficiency and the limited phase deviation, must be used.